Abstract

Human coronaviruses are associated with upper respiratory tract infections that occasionally spread to the lungs and other organs. Although airway epithelial cells represent an important target for infection, the respiratory epithelium is also composed of an elaborate network of dendritic cells (DCs) that are essential sentinels of the immune system, sensing pathogens and presenting foreign antigens to T lymphocytes. In this report, we show that in vitro infection by human coronavirus 229E (HCoV-229E) induces massive cytopathic effects in DCs, including the formation of large syncytia and cell death within only few hours. In contrast, monocytes are much more resistant to infection and cytopathic effects despite similar expression levels of CD13, the membrane receptor for HCoV-229E. While the differentiation of monocytes into DCs in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4 requires 5 days, only 24 h are sufficient for these cytokines to sensitize monocytes to cell death and cytopathic effects when infected by HCoV-229E. Cell death induced by HCoV-229E is independent of TRAIL, FasL, tumor necrosis factor alpha, and caspase activity, indicating that viral replication is directly responsible for the observed cytopathic effects. The consequence of DC death at the early stage of HCoV-229E infection may have an impact on the early control of viral dissemination and on the establishment of long-lasting immune memory, since people can be reinfected multiple times by HCoV-229E.

Cytopathic effects of HCoV-229E on Mo-DCs. (A) Bright-field microscopy of monocytes and Mo-DC cultures either mock treated or infected with HCoV-229E (MOI = 0.05) and then cultured for 24 h. (B) Monocytes (●) and Mo-DCs (▲) were infected with HCoV-229E at MOIs of 0.05, 0.1, and 0.5. Viable cells were enumerated at the indicated time points by trypan blue exclusion (mean of three donors). (C) Detection of the active form of caspase-3 in HCoV-229E-infected Mo-DCs and monocytes by cytometry (the results of one representative experiment out of five are shown). (D) CD13 expression on monocytes and Mo-DCs. Cells were stained with an anti-CD13 MAb (open line) or an isotypic control (closed line) and analyzed by flow cytometry. The data shown are representative of three different donors. (E) Mo-DCs infected with SARS CoV (MOI = 1 for 24 h). The upper part shows a bright-field image of Mo-DCs either mock treated or infected with SARS-CoV. The lower part shows the immunostaining of viral spike glycoprotein and membrane envelope protein (M) (red, anti-spike; green, anti-M; blue, DAPI). The data are representative of three independent experiments.

Susceptibility of monocytes and Mo-DCs to HCoV-229E. (A) Viral spike (S) glycoprotein expression in monocytes and Mo-DC cultures mock treated or infected with HCoV-229E (MOI = 0.05) and then cultured for 10 h (red, anti-spike immunostaining; blue, DAPI staining). (B) Cell surface expression of HCoV-229E spike glycoprotein on mock-treated and HCoV-229E-infected cells (the results of one representative experiment out of three are shown). (C) Kinetics of viral RNA produced in medium from monocytes and Mo-DCs infected with HCoV-229E at MOIs of 0.05, 0.1, and 0.5 (mean of two donors).

Role HCoV-229E cell entry and replication on Mo-DC death. (A) Mo-DCs were incubated or not with anti-CD13 MAb prior to infection with HCoV-229E. Cell death was determined by the observation of cytopathic effects and the formation of syncytia by bright-field microscopy (the results of one representative experiment out of three are shown). (B) Mo-DCs were treated or not with increasing amounts of IFN-β prior to infection with HCoV-229E, and the percentages of cells expressing spike viral protein or activated caspase-3 were determined by flow cytometry (the results of one representative experiment out of three are shown).

Killing of conventional DCs by HCoV-229E. (A) CD34+-DCs, BDCA-1+-DCs, and DC-SIGN+-DCs were either mock treated (open symbols) or infected with HCoV-229E (closed symbols) at different MOIs (0.05, 0.1, and 0.5). Viable cells were enumerated at the indicated time points by trypan blue exclusion (mean of two donors). (B, C, and D) CD34+-DCs were infected with HCoV-229E, cultured for 24 h, and then immunostained for CD13, spike glycoprotein, or active caspase-3 expression. Cells were analyzed by flow cytometry. (E) Mo-DCs were treated with poly-IC, CD40L, or PGE2+TNF-α to induce maturation and then infected with HCoV-229E. The CD80 and HLA-DR expression levels were determined by flow cytometry. (F) Cell death was determined by using Dead/Live staining.

Death of HCoV-229E-infected Mo-DCs is independent of TRAIL, TNF-α, FasL, and caspase activity. (A) Mo-DCs were infected with HCoV-229E and incubated for 24 h in the presence of anti-TRAIL blocking antibodies, TNFR-Fc chimera, or FAS-Fc chimera. Cell death was determined by trypan blue exclusion (the results of one representative experiment out of three are shown). (B) Inhibition of caspase activity does not prevent cell death in Mo-DC cultures. Mo-DCs were preincubated for 24 h in the presence of Z-VAD-FMK, a potent broad-spectrum caspase inhibitor, and then infected by HCoV-229E in the presence of Z-VAD-FMK. After 24 h of culture, cell death was determined by using Dead/Live staining (the results if one representative experiment out of three are shown).

Analysis of monocyte resistance to cytopathic effects and cell death. (A) Mo-DCs were infected with HCoV-229E at an MOI of 0.05 after preincubation for 24 h with culture supernatants from mock-treated monocytes, HCoV-229E-infected monocytes, or UV-treated supernatants from HCoV-229E-infected monocytes. Viable cells were enumerated at the indicated time points by trypan blue exclusion. The results correspond to the mean of three donors. (B) Phenotype analysis of monocytes incubated for 24 h in the presence of GM-CSF and IL-4. Cells were stained with an anti-CD14 MAb or anti-CD11c (open lines) or an isotypic control (closed lines) and analyzed by flow cytometry. The data shown are representative of three different donors. (C) Monocytes become susceptible to HCoV-229E-induced cell death when induced to differentiate into Mo-DCs. Monocytes were treated for 24 h with GM-CSF, IL-4, or both combined and then infected with HCoV-229E. After 24 h, the cells were immunostained for spike glycoprotein and active caspase-3 expression.